Lupin (Lupinus albus), an annual plant belonging to the class of Leguminosae, has been grown since ancient times in the Mediterranean area and in Middle East for its seeds which are used both for alimentary purposes (due to their remarkable protein contents) and in traditional medicine as anthelmintic and antiparasitic agents.
Lupin seeds contain toxic quinolizidine ring alkaloids, such as lupanin, 13-oxy-lupanin, multiflorin and derivatives, and methyl-albin, which are known to exert depressing and paralyzing actions on Central Nervous System. Said alkaloids, which are responsible for the bitter taste of lupin seeds, and occur in large amounts in wild lupin seeds, but in poor amounts in the so-called sweet lupin (Lupinus albus), can be removed by maceration in water.
The prevalent protein fraction in lupin seeds is the globulin one, which accounts for 87% of the total. Said fraction consists of water-insoluble proteins, which are soluble in diluted saline solutions (Duranti et al. Phytochemistry, 20, 2071-2075, 1981). Conglutin gamma accounts for about 6% of the total globulins. The protein apparent molecular weight, as determined by gel-filtration, is approx. 199,000 Da (Duranti et al. in: Lectins: Biology, Biochemistry, Clinical-Biochemistry-Vol. 11 (Van Driesche E, Rougè P, Beeckams S, Bog-Hansen T C eds.) 1997, Textop Publ., Hellerup, Denmark, pp. 881-85, 1997). Conglutin gamma consists of a monomer of apparent molecular weight of 47.000 Da. The reduction of the monomer shows that it consists of two polypeptide chains, with apparent molecular weight of 30.000 Da and 17.000 Da, respectively, linked by a disulfide bridge (Restani et al. Phytochemistry, 20, 2077-2083, 1981).
A tetrameric structure has been suggested for lupin conglutin gamma based on the molecular mass values obtained under native and denaturant conditions. Conglutin gamma light subunit lacks covalently bonded carbohydrates, while the heavy subunit has been found to be glycosylated.
The amino acids composition significantly differs from most lupin spare proteins (Restani et al. 1981, supra). Conglutin gamma contains, in fact, a number of sulfated amino acids and a fair amount of lysine, threonine and trypthophan, and proves very resistant to proteolysis by both endogenous and exogenous proteases (Duranti, Narhung, 30, 271-274, 1986).
The knowledge of the amino acid sequence of the protein (Scarafoni et al., Biochim. Biophys. Acta 1519, 147-151, 2001) allows to exclude any sequence homology with spare proteins, catalytic or structural proteins, also from other sources. Conglutin gamma shows homologies with or similarities to other proteins, such as soy BG7S (70% homology) (Kagawa et al., Febs Letters, 226, 145-149, 1987; Komatsu et al., Biosci. Biotech. Biochem. 58, 1705-1706, 1994) and with EDGP, a glycoprotein from carrot seed (58% homology) (Satoh et al., Planta, 188, 432-438, 1992), whose function has yet to be clarified.
The use of lupin total extract as hypoglycemia was described by Horvath (J. Pharmacol. (Amer.), 38, 303, 1930), which proposed it as a substitute for insulin in mild to medium diabetes mellitus. Subsequently, Clementi and Torrisi (Boll. Soc. It. Biol. Sper., 9, 1004, 1935 e Arch. Fisiol., 34, 290, 1935) identified the hypoglycemizing active ingredient in the alkaloid lupanin, whose effect was however transient.
The hypoglycemizing effect of lupin meal was described also recently in Mario Villaroel et al, Archivos Latinoamericanos de Nutrición, Vol. 46, N. 3, 1996, pp. 234-237), which suggest the use of plums jam containing lupin meal for use as dietetic food for diabetics.
As far as conglutin gamma is concerned, Duranti et al., (Phytochem. 56(6), 529-533, 2001) described its ability to interact with different metals. At pH neutral, conglutin gamma has the highest affinity for Zn2+ ion. Moreover, conglutin gamma is bonded in an affinity chromatography column complexed with Zn2+ and Ni2+; the bonded protein can be eluted using buffering agents at pH below 6 or containing EDTA or imidazole. Conglutin gamma retention curves in metal affinity column are congruent with the titration curve of histidine side group (pKa=6).
However, the use of conglutin gamma for the treatment of type II diabetes has to date not been disclosed.
According to the present invention, it has been found that lupin conglutin gamma as well as proteins showing homology higher than 50% with lupin conglutin gamma, exert remarkable hypoglycemizing action.
Examples of known proteins showing homology higher than 50% with lupin conglutin gamma include soy BG7S (70% homology) (Kagawa et al., Febs Letters, 226, 145-149, 1987; Komatsu et al., Biosci. Biotech. Biochem. 58, 1705-1706, 1994) and EDGP (58% homology) (Satoh et al., Planta, 188, 432-438, 1992).
Conglutin gamma and homologues proteins also proved very powerful in reducing plasma curves after glucose administration in the rat.